The present invention relates to an enzyme feed additive and in particular to such an additive which can decrease the feed conversion ratio of a cereal-based feed and/or increase its digestibility.
Improvements in animal feeds to enable animals to digest them more efficiently are constantly being sought. One of the main concerns is to improve the feed conversion ratio (FCR) of a feed without increasing its cost per unit weight. The FCR is the ratio of the amount of feed consumed relative to the weight gain of an animal. A low FCR indicates that a given amount of feed results in a growing animal gaining proportionately more weight. This means that the animal is able to utilise the feed more efficiently. One way in which the FCR of a feed can be improved is to increase its digestibility.
There are various constraints on the digestibility of the nutritional components of a feed such as its starch, fat, protein and amino acid content. These constraints include:
(i) the viscosity of materials present in the animal""s gut. Such viscosity is due, at least in part, to soluble non-starch polysaccharides such as mixed-linked xcex2-glucans and arabinoxylans;
(ii) entrapment of nutrients within the cell walls of the feed, particularly those of the aleurone layer in cereals. Such entrapment is caused by the high levels of non-starch polysaccharides in the cell walls of cereals which are relatively resistant to break-down by the animal""s digestive system. This prevents the nutrients entrapped within the cells from being nutritionally available to the animal; and
(iii) a deficiency in endogenous enzyme activity, both of the animal and of the gut microbial population particularly in a young animal.
The above problems which interfere with digestibility are particularly noticeable in the case of cereal-based diets, and in particular those having a high barley content.
Due to the problem of poor digestibility of nutrients from the feed, it is normally necessary to formulate feeds to contain higher levels of energy providing materials in order to meet the nutritional demands of animals. Such energy providing materials conventionally include starch, fat, sugars, fibre etc. The requirement of including these energy providing materials, or sources of such materials, in a feed adds a considerable extra cost which is disadvantageous from an economic view point.
In an attempt to solve the problem of poor digestibility of cereal-based feeds, it is known to include enzyme supplements such as xcex2-glucanases or xylanases in animal feeds. For example, WO 91/04673 discloses a feed additive for alleviating malabsorption syndrome in poultry which causes reduced digestion. The additive includes a cellulase and a xylanase. JP-A-60-75238 discloses a feed for domestic animals which contains an enzyme cocktail including protease-, cellulase-, amylase- and lipase-activities. This reference speculates that these various enzyme activities enable fermentation microbes to grow and that these become useful nutritional components of the feed.
Whole cellulase is a mixture of different enzymes which cooperate to hydrolyze cellulose (xcex2-1,4-D-glucan linkages) and/or derivatives thereof (e.g. phosphoric acid swollen cellulose) and give as primary products compounds such as glucose, cellobiose, and cellooligo-saccharides. Whole cellulase is made up of several different enzyme classifications including enzymes having exo-cellobiohydrolase activity, endoglucanase activity and xcex2-glucosidase activity.
For example, the whole cellulase produced by the fungus Trichoderma longibrachiatum comprises two exo-cellobiohydrolases, CBHI and CBHII, at least three endoglucanases, EGI, EGII and EGIII, and at least one xcex2-glucosidase. A representative fermentation from T. longibrachiatum may produce a whole cellulase including by protein weight 45-55% CBHI, 13-15% CBHII, 11-13% EGI, 8-10% EGII, 1-4% EGIII and 0.5-1% BG. However, it should be noted that actual concentrations of a specific cellulase component will vary according to numerous factors, including fermentation conditions, substrate concentrations and strain type. Thus, in a representative fermentation, Trichoderma longibrachiatum produces a whole cellulase having from 58-70% of cellobiohydrolases.
Each endoglucanase of T. longibrachiatum has its own distinct characteristics. Thus, EGI in addition to cellulase activity is known to hydrolyze xylan. EGII and EGIII by comparison do not show significant xylanase activity, at least according to azo-xylan native PAGE overlay. Further, it is known that EGI, EGII and EGV contain structurally distinct cellulose binding domains (CBD""s). On the other hand, EGIII does not appear to contain a structurally distinct binding domain and has been shown to have a lower affinity for crystalline cellulose compared to EGI or EGII.
WO 92/06209 discloses processes for transforming the filamentous fungus Trichoderma reesei (now called xe2x80x9cT. longibrachiatumxe2x80x9d) which involves the steps of treating a T. reesei strain with substantially homologous linear recombinant DNA to permit homologous transformation and then selecting the resulting T. reesei transformants. For instance, transformants are described in which certain targeted genes are deleted or disrupted within the genome and extra copies of certain native genes such as those encoding EGI and EGII are homologously recombined into the strain. It is noted in this reference that cellulase compositions obtained from strains deficient in CBHI and CEHII components are useful as components of a detergent cleaning composition. Such cellulase compositions are of course relatively enriched
When used in vivo, endoglucanases and cellobiohydrolases are considered to act synergistically in the hydrolysis of cellulose to small cello-oligosaccharides (mainly cellobiose), which are subsequently hydrolysed to glucose by the action of xcex2-glucosidase. In addition to hydrolyzing the xcex2-1,4 linkages in cellulose, endo-1,4-xcex2-glucanase (EC 3.2.1.4) will also hydrolyze 1,4 linkages in xcex2-glucans also containing 1,3-linkages. The endoglucanases act on internal linkages to produce cellobiose, glucose and cello-oligosaccharides. The cellobiohydrolases act on the chain ends of cellulose polymers to produce cellobiose as the principal product.
Whole cellulase obtained from T. longibrachiatum has been used in combination with barley in fields such as brewing and in animal nutrition for several years. One of the benefits of adding cellulases to barley-based diets for livestock is to increase the digestibility of various components present in the diet including protein and amino acids. As a result, dietary input costs can be reduced without loss of performance, and excretion of nitrogen in the manure can be significantly reduced. This reduces the environmental impact of intensive livestock farming.
Endosperm cell walls of barley contain a high proportion of high molecular weight, water-soluble mixed-linked xcex2-(1,3)(1,4)-glucans. When solubilised, these poly-saccharides cause an increase in the solution""s viscosity. For example, if barley is fed to broiler chickens, this leads to a relatively high level of viscosity in the region of their gastrointestinal tract, which results in reduced efficiency of digestion and growth depression.
Organisms which produce or express cellulose enzyme complexes often also express xylanase activity. For example, two different xylanase enzymes produced by T. longibrachiatum have been identified. The purification of these two different xylanases, one referred to as high pI xylanase (having a pI of about 9.0) and the other referred to as low pI xylanase (having a pI of about 5.2), as well as the cloning and sequencing of the gene for each xylanase is described in detail in WO 92/06209 and WO 93/24621. FIG. 16 of this document sets out the deduced amino acid sequences for both the low pI and high pI gene products. Example 22 also teaches how to create T. longibrachiatum strains which over-express the low pI and high pI xylanase genes.
As mentioned above, the use of cellulases as an additive to animal feeds is known in the art. Such cellulases of course possess a natural balance between their cellobiohydrolase and endoglucanase contents. AB also mentioned above, in naturally occurring strains of T. longibrachiatum, the CBHs may comprise 58-70% by weight of the cellulase proteins.
The present invention is based upon a study to identify which components of the cellulose proteins are able to improve the nutritional benefits of cereal-based feeds such as those including barley. Specific attention has been paid to the effects of the individual enzymes constituting whole cellulase, and in particular the endoglucanases, on viscosity reduction of soluble mixed-linked xcex2-(1,3) (1,4)-glucans of barley. This is because this is known to be one of the primary modes of action of whole cellulose. The present invention has been made as a result of this research to identify those specific components of the cellulose enzyme system, and their relative amounts, which advantageously improve the feed conversion ratio (FCR) of a cereal-based feed and/or increase its digestibility.
In the description and claims which follow, the following are definitions of some of the technical terms which are employed.
xe2x80x9cFungal cellulasexe2x80x9d means an enzyme composition derived from fungal sources or microorganisms genetically modified so as to incorporate and express all or part of the cellulase genes obtained from a fungal source.
The term xe2x80x9cTrichodermaxe2x80x9d refers to any fungal strain which is or has previously been classified as Trichoderma or which is currently classified as Trichoderma. Such species include Trichoderma longibrachiatum, Trichoderma reesei and Trichoderma viride. 
The term xe2x80x9cEGxe2x80x9d refers to any endoglucanase, for example EGI, EGII, EGIII or EGV produced by T. longibrachiatum, or any derivative of any such endoglucanase which possesses endoglucanase activity.
An EG xe2x80x9cderivativexe2x80x9d includes for example, EGI, EGII, EGIII and EGV from Trichoderma in which there is an addition of one or more amino acids to either or both of the C- and N-terminal ends of the EG, a substitution of one or more amino acids at one or more sites throughout the EG, a deletion of one or more amino acids within or at either or both ends of the EG, or an insertion of one or more amino acids at one or more sites in the EG such that endoglucanase activity is retained in the derivatized EG. The term EG xe2x80x9cderivativexe2x80x9d also includes the core domains of the endoglucanase enzymes that have attached thereto one or more amino acids from the linker regions.
The term xe2x80x9ctruncated cellulasexe2x80x9d, as used herein, refers to a protein comprising a truncated cellulase core of exo-cellobiohydrolase or endoglucanase, for example, EGI, EGII, EGV, CBHI and CBHII, or derivatives of either. EGV is described in Molecular Microbiology, Vol. 13, No. 2 (1994) at pages 219-228. As stated above, many cellulase enzymes, such as EGI, EGII and EGV, are believed to be bifunctional in that they contain regions or domains which are directed toward both catalytic or hydrolytic activity with respect to the cellulose substrate, and also non-catalytic cellulose binding activity. Thus, a truncated cellulase is a cellulase which lacks binding domain cellulose binding activity.
It is believed that the catalytic core and the cellulose binding domain of a cellulose enzyme act together in a synergistic manner to effect efficient hydrolysis of cellulose fibers in a cellulose containing feed. It is further believed that cellulase catalytic activity and cellulose binding activity may be identified as being specific to distinct structural regions, or may be present in the same structural region. For example, as indicated above, many cellulose enzymes, including several of those from T. longibrachiatum are known to incorporate a catalytic core domain subunit which is attached via a linker region to a cellulose binding domain subunit. However, other cellulase enzymes are believed to have a catalytic core domain which is structurally integral to a cellulose binding domain, e.g., the two regions are not separated by a linker and do not represent distinct structural entities. In such a cellulase enzyme, it is believed that a specific peptide or group of related amino acid residues may be responsible for cellulose binding activity. Accordingly, it is within the scope of the present invention that such a binding domain would be altered so as to reduce the cellulose binding activity of the cellulase by, for example, genetic engineering or chemical modification.
A xe2x80x9ctruncated cellulase derivativexe2x80x9d encompasses a truncated cellulase core, as defined herein, wherein there may be an addition or deletion of one or more amino acids to either or both of the C- and N-terminal ends of the truncated cellulose, or a substitution, insertion or deletion of one or more amino acids at one or more sites throughout the truncated cellulase. Derivatives are interpreted to include mutants that preserve their character as truncated cellulase core, as defined below. It is also intended that the term xe2x80x9cderivative of a truncated cellulasexe2x80x9d includes core domains of the exoglucanase or endoglucanase enzymes that have attached thereto one or more amino acids from the linker regions.
A truncated cellulase derivative further refers to a protein substantially similar in structure and biological activity to a truncated cellulose core domain protein, but which has been genetically engineered to contain a modified amino acid sequence. Thus, provided that the two proteins possess a similar activity , they are considered xe2x80x9cderivativesxe2x80x9d as that term is used herein even if the primary structure of one protein does not possess the identical amino acid sequence to that found in the other.
It is contemplated that a truncated cellulase derivative may be derived from a DNA fragment encoding a truncated catalytic core domain which further contains an addition of one or more nucleotides internally or at the 5xe2x80x2 or 3xe2x80x2 end of the DNA fragment, a deletion of one or more nucleotides internally or at the 5xe2x80x2 or 3xe2x80x2 and of the DNA fragment or a substitution of one or more nucleotides internally or at the 5xe2x80x2 or 3xe2x80x2 end of the DNA fragment wherein the functional activity of the catalytic core domain ( truncated cellulase derivative) is retained. Such a DNA fragment (xe2x80x9cvariant DNA fragmentxe2x80x9d) comprising a cellulase catalytic core may further include a linker or hinge DNA sequence or portion thereof which is attached to the core or binding domain DNA sequence at either the 5xe2x80x2 or 3xe2x80x2 end wherein the functional activity of the encoded truncated cellulase core domain (truncated cellulase derivative) is retained.
The term xe2x80x9ctruncated cellulase corexe2x80x9d or xe2x80x9ctruncated cellulase regionxe2x80x9d refers herein to a peptide comprising the catalytic core domain or region of exo-cellobiohydrolase or endoglucanase, for example, EGI, EGII or EGIII or a derivative thereof that is capable of enzymatically cleaving cellulose polymers, including but not limited to pulp or phosphoric acid swollen cellulose. However, a truncated cellulase core will not possess cellulose binding activity attributable to a cellulose binding domain or region. A truncated cellulase core is distinguished from a non-truncated cellulase which, in an intact form, possesses no cellulose binding domain or region. A truncated cellulase core may include other entities which do not include cellulose binding activity attributable to cellulose binding domain or region. For example, the presence of a linker or hinge is specifically contemplated. Similarly the covalent attachment of another enzymatic entity to the truncated cellulase core is also specifically contemplated.
The performance (or activity) of a protein containing a truncated catalytic core or a derivative thereof may be determined by methods well known in the art. (See Wood, T. M. et al. in Methods in Enzymology, Vol. 160, Editors: Wood, W. A. and Kellogg, S. T., Academic Press, pp. 87-116, 1988). For example, such activities can be determined by hydrolysis of phosphoric acid-swollen cellulose and/or soluble oligosaccharides followed by quantification of the reducing sugars released. In this case the soluble sugar products, released by the action of cellobiohydrolase or endoglucanase cellulase core domains or derivatives thereof, can be detected by HPLC analysis or by use of calorimetric assays for measuring reducing sugars. It is expected that these catalytic domains or derivatives thereof will retain at least 10% of the activity exhibited by the intact enzyme when each is assayed under similar conditions and dosed based on similar amounts of catalytic domain protein.
The term xe2x80x9ccellulose binding domainxe2x80x9d refers herein to an amino acid sequence of the endoglucanase comprising the binding domain of an endoglucanase, for example, EGI or EGII, that non-covalently binds to a polysaccharide such as cellulose. It is believed that cellulose binding domains (CBDs) function independently from the catalytic core of the endoglucanase enzyme to attach the protein to cellulose. Truncated endoglucanases used in this invention lack the CBD but include at least the core or catalytic domain.
The term xe2x80x9clinker regionxe2x80x9d or xe2x80x9chinge regionxe2x80x9d refers to the short peptide region that links together the two distinct functional domains of the fungal endoglucanases, i.e., the core domain and the binding domain. These domains in T. longibrachiatum cellulases are linked by a peptide rich in Ser, Thr and Pro.
A xe2x80x9csignal sequencexe2x80x9d refers to any sequence of amino acids bound to the N-terminal portion of a protein which facilitates the secretion of the mature form of the protein outside of the cell. This definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.
The term xe2x80x9chost cellxe2x80x9d means both the cells and protoplasts created from the cells of Trichoderma.
The term xe2x80x9cDNA construct or vectorxe2x80x9d (used interchangeably herein) refers to a vector which comprises one or more DNA fragments or DNA variant fragments encoding any one of the truncated endoglucanases or derivatives described above.
The term xe2x80x9cfunctionally attached toxe2x80x9d means that a regulatory region, such as a promoter, terminator, secretion signal or enhancer region is attached to a structural gene and controls the expression of that gene.
The term xe2x80x9cwhole cellulasexe2x80x9d means the complete cellulase system as produced by a naturally occurring microorganism.
Based upon the above considerations, it is an object of the present invention to provide enzyme-based feed additives which improve the FCR and/or increase the digestibility of a cereal-based feed.
According to one aspect, the present invention provides the use of a composition as a feed additive which comprises one or more endoglucanases, and 0-20% by weight, based upon the content of cellulase proteins in the composition, of a cellobiohydrolase.
As mentioned above, whole cellulase from T. longibrachiatum (i.e. strains occurring naturally) typically contains 58-70% by weight of cellobiohydrolases or more based on the total weight of enzymes having cellulase activity. The composition for use as a feed additive provided by the present invention may be obtained by enriching the content of endoglucanases produced by a suitable microorganism through purification, addition of purified endoglucanase or by adding additional genes to overproduce endoglucanase. In addition, or alternatively, the relative content of cellobiohydrolases produced by the microorganism may be decreased compared to whole cellulase through purification procedures or by modifying or deleting those genes which encode cellobiohydrolase. It is particularly preferred that the feed additive should be free of cellobiohydrolases, so that their content in the additive is 0% by weight.
In a second aspect, the present invention provides an enzyme-based feed additive which comprises at least one of EGIII, EGI which lacks the cellulose binding domain and EGII which lacks the cellulose binding domain, and 0-20% by weight based upon the content of cellulase proteins in the additive, of a cellobiohydrolase.
The production of such structurally modified endoglucanases by genetic engineering techniques is described in detail below.
In a third aspect, the present invention provides an enzyme-based feed additive comprising a cereal-based carrier, one or more endoglucanases, and 0-20% by weight, based upon the content of cellulase proteins in the additive, of a cellobiohydrolase. In such an additive, the cereal-based carrier may be milled wheat, maize or milled soya. Further, the carrier may be a by-product of any of these materials.
Endoglucanases suitable for use in the present invention include those derived from bacterial sources, for example, Bacillus sp., including Bacillus subtilis, Strepromyces sp., Clostridium sp., including Clostridium thermocellum and Clostridium cellulovorans. Alternatively, fungal sources of cellulase are suitable. Suitable fungal sources include Trichoderma sp., Trichoderma longibrachiatum, Trichoderma viride, Trichoderma koningii, Myceliophthora sp., Phanerochaete sp., Schizophyllum sp., Penicillium sp., Aspergillus sp., Geotricum sp., Fusarium sp., Fusarium oxysporum, Humicola sp., Humicola insolens, and Mucor sp., including Mucor miehei. 
Endoglucanase type components may not include components traditionally classified as endoglucanases using activity tests such as the ability of the component (a) to hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (CMC), thereby reducing the viscosity of CMC containing solutions, (b) to readily hydrolyze hydrated forms of cellulose such as phosphoric acid, swollen cellulose (e.g., Walseth cellulose) and hydrolyze less readily the more highly crystalline forms of cellulose (e.g., Avicel, Solkafloc, etc.). On the other hand, it is believed that not all endoglucanase components, as defined by such activity tests, will enhance the nutritional value of feeds. Accordingly, it is more accurate for the purposes herein to define endoglucanase type components as those enzymes which possess feed nutritional enhancement properties comparable to those possessed by the endoglucanase components of Trichoderma longibrachiatum. 
Fungal cellulases can contain more than one endoglucanase type component. The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities, different enzymatic action patterns, etc. The different isoelectric points of the components allow for their separation via ion exchange chromatography and the like. In fact, the isolation of components from different sources is known in the art. See, for example Bjork et al., U.S. Pat. No. 5,120,463; Schulein et al., International Application WO 89/09259; Wood et al., Biochemistry and Genetics of Cellulose Degradation, pp. 31-52 (1988); Wood et al., Carbohydrate Research, Vol. 190, pp. 279-297 (1989); and Schulein, Methods in Enzymology, Vol. 160, pp. 234-242 (1988). The entire disclosure of each of these references is incorpoated herein by reference.
The term xe2x80x9cEGI cellulasexe2x80x9d refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pI) of from about 4.5 to 4.7, and a molecular weight of about 47 to 49 Kdaltons. Preferably, EGI cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride. EGI cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.0, an isoelectric point (pI) of about 4.7 and a molecular weight of about 47 to 49 Kdaltons. EGI cellulase derived from Trichoderma viride has a pH optimum of about 5.0, an isoelectric point (pI) of about 5.3 and a molecular weight of about 50 Kdaltons.
It is noted that EGII has previously been referred to as xe2x80x9cEGIIIxe2x80x9d by some authors but current nomenclature uses the term EGII. In any event the EGII protein differs substantially from the EGIII protein in its molecular weight, pI and pH optimum. The term xe2x80x9cEGII cellulasexe2x80x9d refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 4.0 to 6.0, an isoelectric point (pI) of about 5.5, and a molecular weight of about 35 Kdaltons. Preferably, EGII cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride. 
The term xe2x80x9cEGIII cellulasexe2x80x9d refers to the endoglucanase component derived from Trichoderma spp. characterized by a pH optimum of about 5.0 to 7.0, an isoelectric point (pI) of from about 7.2 to 8.0, and a molecular weight of about 23 to 28 Kdaltons. Preferably, EGIII cellulase is derived from either Trichoderma longibrachiatum or from Trichoderma viride. EGIII cellulase derived from Trichoderma longibrachiatum has a pH optimum of about 5.5 to 6.0, an isoelectric point (pI) of about 7.4 and a molecular weight of about 25 to 28 Kdaltons. EGIII cellulase derived from Trichoderma viride has a pH optimum of about 5.5, an isoelectric point (pI) of about 7.7, and a molecular weight of about 23.5 Kdaltons.
xe2x80x9cExo-cellobiohydrolase type componentsxe2x80x9d (xe2x80x9cCBH type componentsxe2x80x9d) refers to all those cellulase components which exhibit similar feed activity properties to CBHI and CBHII of Trichoderma longibrachiatum. In this regard, when used in combination with EG type components, CBHI and CBHII type components (as defined above) reduce the effectiveness of a cellulase supplement for animal feed in terms of the feed conversion ratio and/or feed digestibility.
Such exo-cellobiohydrolase type components may not include components traditionally classed as exo-cellobiohydrolases using activity tests such as those used to characterize CBHI and CBHII from Trichoderma longibrachiatum. For example, such components (a) are competitively inhibited by cellobiose (K; approximately 1 mM); (b) are unable to hydrolyze to any significant degree substituted celluloses, such as carboxymethylcellulose, etc., and (c) hydrolyze phosphoric acid swollen cellulose and to a lesser degree highly crystalline cellulose. On the other hand, it is believed that some cellulase components which are characterized as CBH components by such activity tests, will enhance the nutritional value of feeds. Accordingly, it is believed to be more accurate for the purposes herein to define such exo-cellobiohydrolases as EG type components because these components possess similar functional properties in animal uses comparable to those of the endoglucanase components of Trichoderma longibrachiatum. 
xe2x80x9cxcex2-glucosidase (BG) componentsxe2x80x9d refer to those components of cellulase which exhibit BG activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides (xe2x80x9ccellobiosexe2x80x9d) and give glucose as the sole product. BG components do not adsorb onto or react with cellulose polymers. Furthermore, such BG components are competitvely inhibited by glucose (Ki approximately 1 mM). While in a strict sense, BG components are not literally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the combined action of CBH components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur. BG components are often characterized by using aryl substrates such as p-nitrophenol-xcex2-D-glucoside (PNPG) and thus are often called aryl-glucosidases. It should be noted that not all aryl-glucosidases are BG components, in that some do not hydrolyze cellobiose.
It is contemplated that the presence or absence of BG components in the cellulase composition can be used to regulate the activity of any CBH components in the composition. Specifically, because cellobiose is produced during cellulose degradation by CBH components, and because high concentrations of cellobiose are known to inhibit CBH activity, and further because such cellobiose is hydrolyzed to glucose by BG components, the absence of BG components in the cellulase composition will xe2x80x9cturn-offxe2x80x9d CBH activity when the concentration of cellobiose reaches inhibitory levels. It is also contemplated that one or more additives (e.g., cellobiose, glucose, etc.) can be added to the cellulase composition to effectively xe2x80x9cturn-offxe2x80x9d, directly or indirectly, some or all of the CBHI type activity as well as other CBH activity. When such additives are employed, the resulting composition is considered to be a composition suitable for use in this invention if the amount of additive is sufficient to lower the CBH type activity to levels equal to or less than the CBH type activity levels achieved by using the cellulase compositions described herein.
On the other hand, a cellulase composition containing added amounts of BG components may increase overall hydrolysis of cellulose if the level of cellobiose generated by the CBH components becomes restrictive of such overall hydrolysis in the absence of added BG components.
Methods to either increase or decrease the amount of BG components in the cellulase composition are disclosed in U.S. Ser. No. 07/807,028 filed Dec. 10, 1991 which is a continuation-in-part of U.S. Ser. No. 07/625,140, filed Dec. 10, 1990 (corresponding to EP-A-0 562 003), all of which are incorporated herein by reference in their entirety.
Fungal cellulases can contain more than one BG component. The different components generally have different isoelectric points which allow for their separation via ion exchange chromatography and the like. Either a single BG component or a combination of BG components can be employed.
In a preferred embodiment, the endoglucanase components suitable for use in the present invention are those which exhibit properties similar to those obtainable from Trichoderma longibrachiatum, i.e., EGI, EGII and EGIII. Thus, the term xe2x80x9cEG type componentsxe2x80x9d refers to all of those cellulase components or combination of components which confer improved properties to feed in a manner similar to the endoglucanase components of Trichoderma longibrachiatum. In this regard, the endoglucanase components of Trichoderma longibrachiatum (specifically, EGI, EGII, EGIII and the like, either alone or in combination) impart characteristics such as improved feed conversion ratio, reduced gut viscosity and improved animal weight gain to animals fed grains treated with them as compared to untreated feed, or feed treated with whole cellulase. Methods for the preparation of EGI, EGII and EGIII are described in detail in WO 92/06209.
It is possible that components other than CBH type components present in the whole cellulase composition may cause undesirable gut viscosity, feed conversion ratio increase and lessened animal weight gain. Therefore, it is contemplated that the use of enriched endoglucanases, such as EGI, EGII or EGIII, may eliminate some or all of the problems which occur when whole cellulase is used.
It has been found that the inclusion of an endoglucanse enriched feed additive in a cereal-based diet of an animal enables the animal to digest the diet more efficiently. This is particularly the case in cereal-based feeds including barley where the presence of the above feed additive improves the feed conversion ratio and/or increases the digestibility of the cereal-based feed. Cereal-based feeds usually include at least 25% by weight of cereal and preferably at least 35% by weight. In addition to or instead of barley, the cereal may include one or more of wheat, triticale, rye and maize.
The endoglucanase enriched feed additives provided by the present invention also enable a conventional cereal-based feed to be modified by reducing its energy, and/or protein, and/or amino acid content whilst simultaneously maintaining the same nutritional levels of energy, protein, and amino acids available to the animal. This means that the amounts of costly energy and protein supplements conventionally included in an animal feed can be reduced as compared to conventional feeds. Energy supplements include fat. Protein supplements include fish-meal, wheat-meal, soya-bean, rapeseed, or canola. This results in a significant reduction in the cost per unit weight of the animal feed without decreasing its nutritional value. Alternatively, or even additionally, the amounts of amino acid supplements can be reduced as compared to conventional feeds which can also result in significant cost savings.
The enzyme feed additive according to the present invention can be prepared in a number of ways. For instance, it can be prepared simply by mixing different enzymes having the appropriate activities to produce an enzyme mix. This enzyme mix can be either mixed directly with a feed, or more conventionally impregnated onto a cereal-based carrier material such as milled wheat, maize or soya flour. A by-product of any of these products may also be used. Such an impregnated carrier constitutes an enzyme feed additive in accordance with the third aspect of the present invention.
As an alternative, a cereal-based carrier formed from e.g. milled wheat or maize can be impregnated either simultaneously or sequentially with enzymes having the appropriate activities. For example, a milled wheat carrier may be sprayed with the one or more endoglucanases. Other enzymes may also be incorporated as appropriate. The carrier material impregnated with these enzymes also constitutes an enzyme feed additive in accordance with the third aspect of the present invention.
The feed additive provided by the present invention may be mixed directly with the animal feed, such as one comprising barley, to prepare the final feed. Alternatively, the feed additive may be mixed with one or more other feed additives such as a vitamin feed additive, a mineral feed additive and an amino acid feed additive. The resulting feed additive including several different types of components can then be mixed in an appropriate amount with the feed.
The resulting cereal-based feed preferably comprises 0.000001-0.1 g/kg of total endoglucanases, more preferably 0.00001-0.01 g/kg and most preferably 0.0001-0.001 g/kg.
The endoglucanases for use in the feed additive of the present invention can be obtained by growing a fungus such as a naturally occurring strain of Trichoderma. Thus, the fungus can be cultivated, after which it is removed from the broth. The cellulase enzyme complex can then be isolated from the broth and separated into its individual components from which the endoglucanases are in turn isolated. This technique is however not so preferred because of the purification steps necessary.
A more preferred method of preparing the enzyme feed additive of the present invention is to construct by genetic manipulation a host microorganism, such as the fungus Trichoderma, which produces the desired enzymes in the appropriate relative amounts. This can be done for instance by increasing the copy number of the gene encoding endoglucanases (e.g. EGI, EGII and/or EGIII) and/or by using a suitably strong promoter in front of any of the above endoglucanase genes. Alternatively or additionally the host strain can be deleted for certain cellulase genes (e.g. those encoding CBHI and/or CBHII). Such procedures are fully explained in the disclosure of WO 92/06209 in the case of transforming T. reesei. 
The enzyme feed additive provided by the present invention may also include other enzymes such as xylanase, protease, xcex1-amylase, glucoamylase, lipase, pectinase, mannanase, xcex1-galactosidase, xcex1-arabinofuranosidase or phytase. Enzymes having the desired activities may for instance be mixed with the endoglucanases used in the present invention either before impregnating these on a cereal-based carrier or alternatively such enzymes may be impregnated simultaneously or sequentially on such a cereal-based carrier. The carrier is then in turn mixed with a cereal-based feed to prepare the final feed. It is also possible to formulate the enzyme feed additive as a solution of the individual enzyme activities and then mix this solution with a feed material pre-formed as pellets or as a mash.
It is also possible to include the enzyme feed additive in the animals"" diet by incorporating it into a second (and different) feed or drinking water which the animal also has access to. Accordingly, it is not essential that the enzyme mix provided by the present invention is incorporated into the cereal-based food itself, although such incorporation forms a particularly preferred aspect of the present invention.
In one preferred embodiment, the xylanase added as an additional enzyme is the high pI xylanase and/or the low pI xylanase obtainable from T. longibrachiatum obtainable by the method of Example 22 of WO 92/06209. It is particularly preferred that the xylanase is the high pI xylanase.
According to a further preferred embodiment, the protease added as an additional enzyme is a subtilisin or mutant thereof derived from the genus Bacillus. Suitable strains of Bacillus include but are not limited to B. amyloliquefaciens, B. lentus, B. licheniformis, B. subtilis, or B. alcalophilus. 
The subtilisin may also be a mutant subtilisin having an amino acid sequence not found in nature but which is derived from a precursor subtilisin by inserting, deleting or replacing one or more different amino acid residues in the precursor subtilisin. Suitable mutant subtilisins are described in EP-A-0 130 756 corresponding to U.S. Pat. No. Re-34686 (including mutations at positions +155, +104, +222, +166, +133, +169, +189, +217, +156, +152); EP-A-0 251 446; WO 91/06637 etc. The most preferred subtilisin is a mutant subtilisin which comprises a substitution at the amino acid residue position equivalent to tyr+217 of B. amyloliquefaciens subtilisin with leucine.
Methods of producing such mutant subtilisins are described in detail in the publications U.S. Pat. No. Re-34606 and EP-A-0 251 446.
The cereal-based animal feeds including the additive of the present invention are suitable for animals such as pigs, ruminants such as sheep and cows, and poultry such as chickens, turkeys, geese and ducks. The feeds though are particularly suitable for poultry and pigs, and in particular broiler chickens.
As previously mentioned, the enzyme feed additive according to the present invention is preferably obtained by growing a genetically modified strain of the fungus Trichoderma. This is because of its well known capacity to secrete whole cellulases in large quantities. This modified strain may be derived from T. longibrachiatum, T. reesei or T. viride. The genome of such strains can be modified to over-express or delete one or more of the enzyme components making up whole cellulase.
Microorganism cultures are grown to a stationary phase, filtered to remove the cells and the remaining supernatant is concentrated by ultrafiltration to obtain the endoglucanase or derivative thereof.
In a particular aspect of the above method, the medium used to cultivate the transformed host cells may be any medium suitable for endoglucanase production in Trichoderma. The endoglucanase or derivative thereof is recovered from the medium by conventional techniques including separation of the cells from the medium by centrifugation, or filtration, precipitation of the proteins in the supernatant or filtrate with salt, for example, ammonium sulphate, followed by chromatography procedures such as ion exchange chromatography, affinity chromatography and the like.
Alternatively, the final protein product may be isolated and purified by binding to a polysaccharide substrate or antibody matrix. The antibodies (polyclonal or monoclonal) may be raised against endoglucanase core domain peptides, or synthetic peptides may be prepared from portions of the core domain and used to raise polyclonal antibodies.
It is further contemplated by the present invention that the DNA fragment or variant DNA fragment encoding the endoglucanase or derivative may be functionally attached to a fungal promoter sequence, for example, the promoter of the cbhl or egl1 gene. Also contemplated by the present invention is manipulation of the Trichoderma strain via transformation such that a DNA fragment encoding an endoglucanase or derivative thereof is inserted within the genome. It is also contemplated that more than one copy of an endoglucanase DNA fragment or DNA variant fragment may be recombined into the strain.
A selectable marker must first be chosen so as to enable detection of the transformed fungus. Any selectable marker gene which is expressed in Trichoderma can be used in the present invention so that its presence in the transformants will not materially affect the properties thereof. The selectable marker can be a gene which encodes an assayable product. The selectable marker may be a functional copy of a Trichoderma gene which if lacking in the host strain results in the host strain displaying an auxotrophic phenotype.
The host strains used could be derivatives of Trichoderma which lack or have a non-functional gene or genes corresponding to the selectable marker chosen. For example, if the selectable marker of pyr4 is chosen, then a specific pyr derivative strain is used as a recipient in the transformation procedure. Other examples of selectable markers that can be used in the present invention include the Trichoderma genes equivalent to the Aspergillus nidulans genes argb, trpc, niaD and the like.
The corresponding recipient strain must therefore be a derivative strain such as argBxe2x88x92, trpCxe2x88x92, niaDxe2x88x92, and the like.
The strain is derived from a starting host strain which is any Trichoderma strain. However, it is preferable to use a T. longibrachiatum cellulase over-producing strain such as RL-P37, described by Sheir-Neiss et al. in Appl. Microbiol. Biotechnology, 20 (1984) pp. 46-53, since this strain secretes elevated amounts of cellulase enzymes. This strain is then used to produce the derivative strains used in the transformation process.
The derivative strain of Trichoderma can be prepared by a number of techniques known in the art. An example is the production of pyr4xe2x88x92 derivative strains by subjecting the strains to fluoroorotic acid (FOA). The pyr4 gene encodes orotidine-5xe2x80x2-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. Strains with an intact pyr4 gene grow in a medium lacking uridine but are sensitive to fluoroorotic acid. It is possible to select pyr4xe2x88x92 derivative strains which lack a functional orotidine monophosphate decarboxylase enzyme and require uridine for growth by selecting for FOA resistance. Using the FOA selection technique, it is also possible to obtain uridine requiring strains which lack a functional orotate pyrophosphoribosyl transferase. It is possible to transform these cells with a functional copy of the gene encoding this enzyme (Berges and Barreau, 1991, Curr. Genet. 19 pp359-365). Since it is easy to select derivative strains using the FOA resistance technique in the present invention, it is preferable to use the pyr4 gene as a selectable marker.
In a preferred embodiment of the present invention, Trichoderma host cell strains are deleted of one or more cellobiohydrolase genes prior to introduction of a DNA construct or plasmid containing the DNA fragment encoding the endoglucanase of interest. It is preferable to express an endoglucanase, derivative thereof or covalently linked endoglucanase domain derivative in a host that is missing one or more cellobiohydrolase genes in order to simplify the identification and subsequent purification procedures. Any gene from Trichoderma which has been cloned can be deleted such as cbhl or cbh2.
The desired gene that is to be deleted from the transformant is inserted into a plasmid by methods known in the art. This plasmid is selected such that unique restriction enzyme sites are present therein to enable the fragment of Trichoderma DNA to be subsequently removed as a single linear piece. The plasmid containing the gene to be deleted or disrupted is then cut at appropriate restriction enzyme site(s), internal to the coding region, the gene coding sequence or part thereof may be removed therefrom and the selectable marker (e.g. pry 4) inserted. Flanking DNA sequences from the locus of the gene to be deleted or disrupted, preferably between about 0.5 to 2.0 kb, remain on either side of the selectable marker gene.
A single DNA fragment containing the deletion construct is then isolated from the plasmid and used to transform the appropriate pyrxe2x88x92 Trichoderma host. Transformants are selected based on their ability to express the pyr4 gene product and thus complement the uridine auxotrophy of the host strain. Southern blot analysis is then carried out on the resultant transformants to identify and confirm a double cross-over integration event which replaces part or all of the coding region of the gene to be deleted with the pyr4 selectable markers.
Although specific plasmid vectors are described above, the present invention is not limited to the production of these vectors. Various genes can be deleted and replaced in the Trichoderma strain using the above techniques. Any available selectable markers can be used, as discussed above. Potentially any Trichoderma gene which has been cloned, and thus identified, can be deleted from the genome using the above-described strategy.
The expression vector of the present invention carrying the inserted DNA fragment or variant DNA fragment encoding the endoglucanase or derivative thereof of the present invention may be any vector which is capable of replicating autonomously in a given host organism, typically a plasmid. In preferred embodiments two types of expression vectors for obtaining expression of genes or truncations thereof are contemplated. The first contains DNA sequences in which the promoter, gene coding region, and terminator sequence all originate from the gene to be expressed. Gene truncation if required is obtained by deleting away the undesired DNA sequences (coding for unwanted domains) to leave the domain to be expressed under control of its own transcriptional and translational regulatory sequences. A selectable marker is also contained on the vector allowing the selection for integration into the host of multiple copies of the novel gene sequences.
For example, a DNA construct which can be termed pEGID3xe2x80x2pyr contains the EGI cellulase core domain under the control of the EGI promoter, terminator, and signal sequences. The 3xe2x80x2 end on the EGI coding region containing the cellulose binding domain has been deleted. The plasmid also contains the pyr4 gene for the purpose of selection.
The second type of expression vector is preassembled and contains sequences required for high level transcription and a selectable marker. It is contemplated that the coding region for a gene or part thereof can be inserted into this general purpose expression vector such that it is under the transcriptional control of the expression cassette""s promoter and terminator sequences.
For example, pTEX is such a general purpose expression vector. Genes or part thereof can be inserted downstream of the strong CBHI promoter.
In the vector, the DNA sequence encoding the endoglucanase should be operably linked to transcriptional and translational sequences, i.e., a suitable promoter sequence and signal sequence in reading frame to the structural gene. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The signal peptide provides for extracellular expression of the endoglucanase or derivatives thereof. The DNA signal sequence is preferably the signal sequence naturally associated with the truncated gene to be expressed, however the signal sequence from any endoglucanase is contemplated in the present invention.
The procedures used to ligate the DNA sequences coding for the truncated endoglucanases or derivatives thereof with the promoter, and insertion into suitable vectors containing the necessary information for replication in the host cell are well known in the art.
The DNA vector or construct described above may be introduced in the host cell in accordance with known techniques such as transformation, transfection, microinjection, microporation, biolistic bombardment and the like.
In a preferred embodiment of the present invention, the modified strain is derived from Trichoderma sp. containing deleted or disrupted genes for CBHI and/or CBHII thereby being unable to produce catalytically active cellobiohydrolase. The cellulase enzymes produced by such an organism will be enriched in endoglucanases and include no more than 20% cellobiohydrolases based upon the combined weight of cellulase proteins which it produces. It is particularly preferred that the modified strain is unable to produce catalytically active CBHI as this enzyme forms the greatest proportion o any component of whole cellulase from Trichoderma sp. In instances where only production of EGIII is desired, it is further preferred that such a modified strain contains deleted or disrupted genes for EGI and EGII so as to be unable to produce catalytically active EGI and/or EGII.
Alternatively, the modified strain can additionally contain recombinant DNA allowing expression and secretion of truncated catalytic cores of either EGI or EGII. While not wishing to be bound by theory, it is believed that the presence of a cellulose binding domain on a cellulase may be responsible for certain undesirable properties observed when animals are fed feed supplemented with cellulase, e.g. increased gut viscosity. Accordingly, by removing the cellulose binding domain and retaining an intact cellulase core, it is possible to limit or eliminate these properties.